The Rep proteins of Adeno-associated virus type 2 (AAV) have potential as anti-cancer and anti-HIV therapeutics. The AAV rep gene open reading frame encodes four overlapping Rep proteins. Rep68 and Rep78 bind to the AAV genome's inverted terminal repeats, have enzymatic activities which are necessary for AAV DNA replication, regulate AAV gene expression, inhibit HIV-1 replication, are required for preferential integration of the AAV genome into a region of human chromosome 19, and inhibit cell division. Wild-type and mutant Rep proteins were expressed from the HIV- 1 long terminal repeat promoter in human 293 cell transient transfections or as maltose binding protein fusions in Escherichia coli. The ability of Rep proteins to down-regulate AAV promoters is optimal when the promoter contains a Rep binding site and the Rep proteins contain a wild- type ATP binding site. Although Rep68 binding to DNA is enhanced by the presence of ATP, wild-type Rep68 can still down-regulate, to a lesser degree, promoters which show no stable Rep binding in the presence or absence of ATP. Based on the consensus binding sequence and electrophoretic mobility shift assays, binding sites for Rep proteins have been identified within several human genes, some of which are known to be involved in the regulation of cell proliferation. A study has been initiated to determine the impact of Rep proteins on the expression of these genes. We have observed a correlation between the ATP-dependent helicase activity of wild-type and mutant Rep proteins and their ability to efficiently down-regulate the p5 and p19 promoters of AAV. Further studies have revealed that mutant Rep proteins with a defect in the ATP binding site are dominant-negative for p5 and p19 down-regulation, DNA helicase activity, and RNA/DNA helicase activity. These observations are consistent with a model in which Rep proteins function as multimers and can down-regulate genes via their helicase activities.